Impact of linker histone in the formation of ambochlorin-calf thymus DNA complex: Multi-spectroscopic, stopped-flow, and molecular modeling approaches

Document Type : Original Article


1 Department of Biology, Faculty of Sciences, Mashhad Branch, Islamic Azad University, Mashhad, Iran

2 Medical Chemistry Department, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s): This study aimed to evaluate the role of the linker histone (H1) in the binding interaction between ambochlorin (Amb), and calf thymus DNA (ctDNA) as binary and ternary systems. 
Materials and Methods: The project was accomplished through the means of absorbance, fluorescence, stopped-flow circular dichroism spectroscopy, viscosity, thermal melting, and molecular modeling techniques.
Results: Spectroscopic analysis revealed that although Amb was strongly bound to both ctDNA and ctDNA-H1, it showed a greater tendency to ctDNA in the presence of the linker histone. The obtained thermodynamic parameters revealed that both Amb-ctDNA and Amb-ctDNA-H1 interactions were spontaneous, endothermic, and entropy-favored, and hydrophobic interactions played the main role in the formation and stabilization of complexes. Analysis of the stopped-flow circular dichroism results revealed that the binding process of Amb-ctDNA and Amb-ctDNA-H1 required a time of more than 150 milliseconds to complete. Moreover, Amb-ctDNA complex formation was marginally decelerated in the presence of the linker histone. The docking results suggested that the presence of the  linker histone may alter the binding sites of Amb from ctDNA minor grooves to major grooves. 
Conclusion: All quenching processes were governed by a dynamic mechanism. Additionally, Amb did not stabilize or induce considerable conformational changes in ctDNA and ctDNA-H1 complex upon binding. In silico molecular docking results confirmed that Amb was bound to the double-helical ctDNA and ctDNA-H1 via ctDNA grooves. In summary, some binding properties of the interactions between Amb and ctDNA change in the presence of the linker histone.


1. Shi J-H, Chen J, Wang J, Zhu Y-Y. Binding interaction between sorafenib and calf thymus DNA: spectroscopic methodology, viscosity measurement and molecular docking. Spectrochim Acta A Mol Biomol Spectrosc 2015; 136:443-450.
2. Berthet N, Boudali A, Constant JF, Decout JL, Demeunynck M, Fkyerat A, et al. Design of molecules that specifically recognize and cleave apurinic sites in DNA. J Mol Recognit 1994; 7:99-107.
3. Nieto Moreno N, Giono LE, Botto C, Adrián E, Muñoz MJ, Kornblihtt AR. Chromatin, DNA structure and alternative splicing. FEBS Letters 2015; 589:3370-3378.
4.Anwer R, Ahmad N, Al Qumaizi KI, Al Khamees OA, Al Shaqha WM, Fatma T. Interaction of procarbazine with calf thymus DNA-a biophysical and molecular docking study. J Mol Recognit 2017; 30:1-6.
5. Marko JF. Biophysics of protein–DNA interactions and chromosome organization. Physica A: Statistical Mechanics and its Applications 2015; 418:126-153.
6. Rosenfeld JA, Wang Z, Schones DE, Zhao K, DeSalle R, Zhang MQ. Determination of enriched histone modifications in non-genic portions of the human genome. BMC Genomics 2009; 10:1-11.
7. Chandrasekaran S, Sameena Y, Enoch IV. Tuning the binding of coumarin 6 with DNA by molecular encapsulators: effect of beta-cyclodextrin and C-hexylpyrogallol[4]arene. J Mol Recognit 2014; 27:640-652.
8. Wu H-C, Chang D-K, Huang C-T. Targeted therapy for cancer. J Cancer Mol 2006; 2:57-66.
9. Taşkın A, Tarakçıoğlu M, Ulusal H, Örkmez M, Taysi S. Idarubicin-bromelain combination sensitizes cancer cells to conventional chemotherapy. Iran J Basic Med Sci 2019;22:10:1-8.
10. Rehman SU, Sarwar T, Husain MA, Ishqi HM, Tabish M. Studying non-covalent drug–DNA interactions. Arch Biochem Biophys 2015; 576:49-60.
11. Boulikas T, Vougiouka M. Cisplatin and platinum drugs at the molecular level. Oncolo Rep 2003; 10:1663-1682.
12. Ozdemir F, Sever A, Öğünç Keçeci Y, Incesu Z. Resveratrol increases the sensitivity of breast cancer MDA-MB-231 cell line to cisplatin by regulating intrinsic apoptosis. Iran J Basic Med Sci 2021;24:1:66-72.
13. Gunasekaran S, Kumaresan S, Balaji RA, Anand G, Seshadri S. Vibrational spectra and normal coordinate analysis on structure of chlorambucil and thioguanine. Paramna J Phys 2008; 71:1291-1300.
14. Charak S, Shandilya M, Tyagi G, Mehrotra R. Spectroscopic and molecular docking studies on chlorambucil interaction with DNA. Int J Biol Macromol 2012; 51:406-411.
15. Farmer P. Metabolism and reactions of alkylating agents. Pharm& Ther 1987; 35:301-358.
16. Rehman SU, Sarwar T, Ishqi HM, Husain MA, Hasan Z, Tabish M. Deciphering the interactions between chlorambucil and calf thymus DNA: a multi-spectroscopic and molecular docking study. Arch Biochem Biophys 2015; 566:7-14.
17. Parker LJ, Ciccone S, Italiano LC, Primavera A, Oakley AJ, Morton CJ, et al. The anti-cancer drug chlorambucil as a substrate for the human polymorphic enzyme glutathione transferase P1-1: kinetic properties and crystallographic characterisation of allelic variants. J Mol Biol 2008; 380:131-144.
18. Brooks WH, Renaudineau Y. Epigenetics and autoimmune diseases: the X chromosome-nucleolus nexus. Front In Gene 2015; 6:1-20.
19.Eibl C, Hessenberger M, Wenger J, Brandstetter H. Structures of the NLRP14 pyrin domain reveal a conformational switch mechanism regulating its molecular interactions. Acta Crystallogr D Biol Crystallogr 2014; 70:2007-2018.
20. Desilets DJ, Kissinger PT, Lytle FE. Improved method for determination of Stern-Volmer quenching constants. Anal Chem 1987; 59:1244-1246.
21. Guo X-J, Hao A-J, Han X-W, Kang P-L, Jiang Y-C, Zhang X-J. The investigation of the interaction between ribavirin and bovine serum albumin by spectroscopic methods. Mol Biol Rep 2011; 38:4185-4192.
22. Lindon JC, Tranter GE, Koppenaal D. Encyclopedia of spectroscopy and spectrometry: Academic Press 2016.
23. Shi J-H, Chen J, Wang J, Zhu Y-Y, Wang Q. Binding interaction of sorafenib with bovine serum albumin: Spectroscopic methodologies and molecular docking. Spectrochim Acta A Mol Biomol Spectrosc 2015; 149:630-637.
24. Perozzo R, Folkers G, Scapozza L. Thermodynamics of protein–ligand interactions: History, presence, and future aspects. J Recept Signal Transduct 2004; 24:1-52.
25. Du X, Li Y, Xia Y-L, Ai S-M, Liang J, Sang P, et al. Insights into protein–ligand interactions: mechanisms, models, and methods. Int J Mol Sci 2016; 17:144-178.
26. Ross PD, Subramanian S. Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 1981; 20:3096-3102.
27. Haq I. Thermodynamics of drug–DNA interactions. Arch Biochem Biophys 2002; 403:1-15.
28. Collings PJ, Gibbs EJ, Starr TE, Vafek O, Yee C, Pomerance LA, et al. Resonance light scattering and its application in determining the size, shape, and aggregation number for supramolecular assemblies of chromophores. J Phys Chem B 1999; 103:8474-8481.
29. Li L, Pan Q, Wang YX, Song GW, Xu ZS. Study on the binding equilibrium between surfactant FC95 and DNA by resonance light-scattering technique. Appl Surf Sci 2011; 257:4547-4551.
30. Qiao C, Bi S, Sun Y, Song D, Zhang H, Zhou W. Study of interactions of anthraquinones with DNA using ethidium bromide as a fluorescence probe. Spectrochim Acta A Mol Biomol Spectrosc 2008; 70:136-143.
31. Waring M. Complex formation between ethidium bromide and nucleic acids. J Mol Biol 1965; 13:269-282.
32. Olmsted III J, Kearns DR. Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids. Biochem 1977; 16:3647-3654.
33. Bi S, Qiao C, Song D, Tian Y, Gao D, Sun Y, et al. Study of interactions of flavonoids with DNA using acridine orange as a fluorescence probe. Sens Actuators B Chem 2006; 119:199-208.
34. Patel DJ. Nuclear magnetic resonance studies of drug-nucleic acid interactions at the synthetic DNA level in solution. Acc Chem Res 1979; 12:118-125.
35. Kumar C, Turner R, Asuncion E. Groove binding of a styrylcyanine dye to the DNA double helix: the salt effect. J Photochem Photobiol A Chem 1993; 74:231-238.
36. Kelly JM, Tossi AB, McConnell DJ, OhUigin C. A study of the interactions of some polypyridylruthenium (II) complexes with DNA using fluorescence spectroscopy, topoisomerisation and thermal denaturation. Nucleic Acids Res 1985; 13:6017-6034.
37. Wheate NJ, Brodie CR, Collins JG, Kemp S, Aldrich-Wright JR. DNA intercalators in cancer therapy: organic and inorganic drugs and their spectroscopic tools of analysis. Mini Rev Med Chem 2007; 7:627-648.
38. Afrin S, Rahman Y, Sarwar T, Husain MA, Ali A, Tabish M. Molecular spectroscopic and thermodynamic studies on the interaction of anti-platelet drug ticlopidine with calf thymus DNA. Spectrochim Acta A Mol Biomol Spectrosc 2017; 186:66-75.
39. Chen CB, Chen J, Wang J, Zhu YY, Shi JH. Combined spectroscopic and molecular docking approach to probing binding interactions between lovastatin and calf thymus DNA. Lumin 2015; 30:1004-1010.
40. Scott RA, Lukehart CM. Applications of physical methods to inorganic and bioinorganic chemistry: John Wiley & Sons 2013.
41. Chen C, Li M, Xing Y, Li Y, Joedecke C-C, Jin J, et al. Study of pH-induced folding and unfolding kinetics of the DNA i-motif by stopped-flow circular dichroism. Langmuir 2012; 28:17743-17748.
42. Zhang G, Fu P, Pan J. Multispectroscopic studies of paeoniflorin binding to calf thymus DNA in vitro. J Lumine 2013; 134:303-309.
43. Alsaif NA, Wani TA, Bakheit  AH, Zargar S. Multi-spectroscopic investigation, molecular docking and molecular dynamic simulation of competitive interactions between flavonoids (quercetin and rutin) and sorafenib for binding to human serum albumin. Int J Biol Macromole 2020;15;165:2451-2461.
44.Fu X, Belwal T, He Y, Xu Y, Li L, Luo Z. Interaction and binding mechanism of cyanidin-3-O-glucoside to ovalbumin in varying pH conditions: A spectroscopic and molecular docking study. Food Chem 2020;1;320:126616.
45. Simunkova M, Steklac M, Malcek M. Spectroscopic, computational and molecular docking study of Cu (ii) complexes with flavonoids: from cupric ion binding to DNA intercalation. New J Chem  2021;45:10551-10972.
46. Devanesan, S., AlSalhi, M.S., Masilamani, V., Alqahtany, F., Rajasekar, A., Alenazi, A. and Farhat, K.,. Fluorescence spectroscopy as a novel technique for premarital screening of sickle cell disorders. Photodiagnosis Photodyn Ther 2021; 34: 102276.
47. Lima, E.C., Hosseini-Bandegharaei, A., Moreno-Piraján, J.C. and Anastopoulos, I..A critical review of the estimation of the thermodynamic parameters on adsorption equilibria. Wrong use of equilibrium constant in the Van’t Hoof equation for calculation of thermodynamic parameters of adsorption. J Mol Liq 2019; 273, pp.425-434.
48. Ghosal PS, Gupta AK. Determination of thermodynamic parameters from Langmuir isotherm constant-revisited. J Mol Liq  2017;1;225:137-146.
49. Pirdadeh-Beiranvand M, Afkhami A, Madrakian T. Ag nanoparticles for determination of bisphenol A by resonance light-scattering technique. Journal of the Iranian Chemical Society 2018 15:1527-1534.
50. Ezhuthupurakkal PB, Polaki LR, Suyavaran A, Subastri A, Sujatha V, Thirunavukkarasu C. Selenium nanoparticles synthesized in aqueous extract of Allium sativum perturbs the structural integrity of Calf thymus DNA through intercalation and groove binding. Mater Sci Eng 2017; C;74:597-608.
51. Keswani N, Panicker A. Binding behaviour of aminoglycoside drug kanamycin with calf thymus DNA: Thermodynamic, spectroscopic and molecular modelling studies. Thermochim Acta 2021; 1;697:178856.
52. Kou SB, Lou YY, Zhou KL, Wang BL, Lin ZY, Shi JH. In vitro exploration of interaction behavior between calf thymus DNA and fenhexamid with the help of multi-spectroscopic methods and molecular dynamics simulations. J Mol Liq 2019;15;296:112067.
53. Liu C, Cheng F, Yang X. Inactivation of soybean trypsin inhibitor by epigallocatechin gallate: Stopped-flow/fluorescence, thermodynamics, and docking studies. J Agric Food Chem 2017; 1;65:921-929.